This invention relates in general to plant molecular biology and plant genetic engineering for herbicide resistance and, more particularly, to a novel glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase from Eleusine indica. Plant genetic engineering methods can be used to transfer the glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase gene isolated and purified from Eleusine indica into crop and ornamental plants of economic importance.
N-phosphonomethylglycine, also known as glyphosate, is a well known herbicide that has activity on a broad spectrum of plant species. Glyphosate is the active ingredient of Roundup(copyright) (Monsanto Co.), a safe herbicide having a desirably short half life in the environment. When applied onto a plant surface, glyphosate moves systemically through the plant. Glyphosate is toxic to plants by inhibiting the shikimic acid pathway that provides a precursor for the synthesis of aromatic amino acids. Specifically, glyphosate affects the conversion of phosphoenolpyruvate and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (hereinafter referred to as EPSP synthase or EPSPS). For purposes of the present invention, the term xe2x80x9cglyphosatexe2x80x9d should be considered to include any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in planta.
Through plant genetic engineering methods, it is possible to produce glyphosate tolerant plants by inserting into the plant genome a DNA molecule that causes the production of higher levels of wild-type EPSPS (Shah et al., Science 233:478-481 (1986). Glyphosate tolerance can also be achieved by the expression of EPSPS variants that have lower affinity for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (U.S. Pat. No. 4,940,835, U.S. Pat. No. 5,094,945, U.S. Pat. No. 5,633,435). Enzymes that degrade glyphosate in the plant tissues (U.S. Pat. No. 5,463,175) are also capable of conferring cellular tolerance to glyphosate. Such genes, therefore, allow for the production of transgenic crops that are tolerant to glyphosate, thereby allowing glyphosate to be used for effective weed control with minimal concern of crop damage. For example, glyphosate tolerance has been genetically engineered into corn (U.S. Pat. No. 5,554,798), wheat (Zhou et al. Plant Cell Rep. 15:159-163 (1995), soybean (WO 9200377) and canola (WO 9204449).
Variants of the wild-type EPSPS enzyme are glyphosate-resistant as a result of alterations in the EPSPS amino acid coding sequence (Kishore et al., Annu. Rev. Biochem. 57:627-663 (1988); Schulz et al., Arch. Microbiol. 137:121-123 (1984); Sost et al., FEBS Lett. 173:238-241 (1984); Kishore et al., In xe2x80x9cBiotechnology for Crop Protectionxe2x80x9d ACS Symposium Series No. 379. Eds. Hedlin et al., 37-48 (1988). These variants typically have a higher Ki for glyphosate than the wild-type EPSPS enzyme that confers the glyphosate-tolerant phenotype, but these variants are also characterized by a high Km for PEP that makes the enzyme kinetically less efficient. For example, the apparent Km for PEP and the apparent Ki for glyphosate for the native EPSPS from E. coli are 10 xcexcM and 0.5 xcexcM while for a glyphosate-resistant isolate having a single amino acid substitution of an alanine for the glycine at position 96 these values are 220 xcexcM and 4.0 mM, respectively. A number of glyphosate-resistant plant variant EPSPS genes have been constructed by mutagenesis.
A variety of native and variant EPSPS enzymes have been expressed in transgenic plants in order to confer glyphosate tolerance (Singh, et al., In xe2x80x9cBiosynthesis and Molecular Regulation of Amino Acids in Plantsxe2x80x9d, Amer Soc Plant Phys. Pubs (1992). Examples of some of these EPSP Synthases and methods for preparing transgenic plants resistant to glyphosate include those described and/or isolated in accordance with U.S. Pat. Nos. 4,940,835, 4,971,908, 5,145,783, 5,188,642, 5,310,667, 5,312,910, and 6,40,497. They can also be derived from a structurally distinct class of non-homologous EPSPS genes, such as the naturally occurring class II EPSPS genes isolated from Agrobacterium sp. strain CP4 as described in U.S. Pat. No. 5,633,435 and U.S. Pat. No. 5,627,061.
Eleusine indica is commonly referred to as xe2x80x9cgoose grassxe2x80x9d and may also be known as xe2x80x9cyard grassxe2x80x9d. It is a common monocotyledonous plant found world wide. As a member of the Poaceae family, the grass family, it is related to many well known crop plants. Eleusine indica is most closely related to the millets, that include Sorghum bicolor (sorghum or great millet), Zea mays (maize), Pennisetum americanum (pearl millet), Eleusine coracana (finger millet), Setaria italica (foxtail millet), Paspalum scrobiculatum (kodo millet), Echinochloa frumentacea (barnyhard millet) and Eragrostis tef (teff) (Chennaveeraiah et al., In xe2x80x9cChromosome engineering in plants: genetics, breeding and evolutionxe2x80x9d, Cytogenetics of Minor Millets, in Tsuchiya et al., eds Elsevier Sci Pub Amsterdam, 613-627 (1991). Eleusine indica has been shown to hybridize with Eleusine coracana (finger millet), an important cultivated millet of India and East Africa (Chennaveeraiah et al., Euphytica 2-3:489-495, (1974). Classical plant breeding methods can be used to transfer the genes and traits of interest from Eleusine indica into agronomic crop plants within the family Poaceae.
In its broadest sense, the present invention herein provides a method for plant tolerance to glyphosate herbicide by the expression of an isolated DNA molecule encoding a naturally occurring glyphosate resistant EPSPS enzyme. The enzyme and the DNA is isolated from Eleusine species, more particularly Eleusine indica (E. indica).
In the first aspect of the present invention described herein provides a method to cause plants to be tolerant to glyphosate herbicide by the insertion of a recombinant DNA molecule into the nuclear genome of a plant cell, the recombinant DNA molecule comprising:
a promoter that functions in plant cells to cause the production of an RNA molecule; operably linked to,
a DNA molecule transcribing an RNA encoding for a chloroplast transit peptide and a E. indica glyphosate resistant EPSPS enzyme; operably linked to,
a 3xe2x80x2 non-translated region that functions in plant cells to cause the polyadenylation of the 3xe2x80x2 end of the RNA molecule.
Typically, the promoter used in the DNA molecule is expressed in a constitutive fashion. Examples of suitable promoters that function effectively in this capacity include cauliflower mosaic virus 19S promoter, cauliflower mosaic virus 35S promoter, figwort mosaic virus 34S promoter, sugarcane bacilliform virus promoter, commelina yellow mottle virus promoter, small subunit of ribulose-1,5-bisphosphate carboxylase promoter, rice cytosolic triosephosphate isomerase promoter, adenine phosphoribosyltransferae promoter, rice actin 1 promoter, maize ubiquitin promoter, mannopine synthase promoter and octopine synthase promoter. A Promoter may also comprise leader sequences and intron sequences useful in the invention.
A DNA molecule that encodes a chloroplast transit peptide sequence can be isolated from EPSPS genes purified from various plant species including E. indica as well as from various plant genes whose protein products have been shown to be transported into the chloroplast.
A DNA molecule that encodes a glyphosate resistant EPSPS enzyme isolated from Eleusine species, more particularly from E. indica, comprising SEQ ID NO:7 is an object of the invention and a DNA molecule substantially homologous to the DNA molecule isolated from E. indica or a portion thereof identified as SEQ ID NO:6.
The 3xe2x80x2 non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3xe2x80x2 untranslated, the 3xe2x80x2 untranslated region from pea small subunit Rubisco gene, the wheat heat shock protein 17.9 3xe2x80x2 untranslated region, the 3xe2x80x2 untranslated region from soybean 7S seed storage protein gene are commonly used in this capacity.
The invention also relates to a glyphosate tolerant transgenic crop plant cell, a glyphosate tolerant crop plant and crop plant parts, crop seeds and progeny thereof comprising the recombinant DNA molecule of the present invention.
A DNA molecule that encodes a naturally occurring plant derived glyphosate resistant EPSPS enzyme, wherein the glyphosate resistant EPSPS enzyme has a Km for phosphoenolpyruvate (PEP) of less than 10 xcexcM. More preferably, a DNA molecule that encodes a naturally occurring plant derived glyphosate resistant EPSPS enzyme wherein the glyphosate resistant EPSPS enzyme has a Km for PEP of less than 10 xcexcM and the Km for PEP is not more than about 2xc3x97 of the naturally occurring plant derived glyphosate sensitive EPSPS enzyme.
A DNA molecule that encodes a naturally occurring glyphosate resistant EPSPS enzyme derived from Eleusine species, wherein the glyphosate resistant EPSPS enzyme has a Km for phosphoenolpyruvate (PEP) of less than 10 xcexcM. More preferably, a DNA molecule that encodes a naturally occurring glyphosate resistant EPSPS enzyme derived from Eleusine species, wherein the glyphosate resistant EPSPS enzyme has a Km for PEP of less than 10 xcexcM and the Km for PEP is not more than about 2xc3x97 of the naturally occurring plant derived glyphosate sensitive EPSPS enzyme.
A DNA molecule that encodes a naturally occurring glyphosate resistant EPSPS enzyme derived from E. indica, wherein the naturally occurring glyphosate resistant EPSPS enzyme amino acid sequence has been modified by amino acid substitutions selected from the group consisting of threonine to isoleucine at amino acid position 103 and glycine to alanine at amino acid position 102.
The invention also relates to the homologous genetic elements regulating expression of the E. indica glyphosate resistant EPSPS gene. These elements include but are not limited to the DNA sequences of a promoter, a 5xe2x80x2 untranslated region, a chloroplast transit peptide, an intron, and a 3xe2x80x2 untranslated region of E. indica EPSPS glyphosate resistance gene. A DNA molecule that encodes a glyphosate resistant EPSPS enzyme purified from the genome of Eleusine species, more particularly from E. indica glyphosate resistant biotype provided by the ATCC deposit #PTA-2177 is an object of the invention.